Identification of 14-hydroxy-4,14-retro-retinol as an in vivo metabolite of vitamin A.

Retinol (vitamin A alcohol) undergoes extensive metabolism in vertebrates. We report here (i) the identification of a yet undescribed in vivo metabolite of retinol as 14-hydroxy-4,14-retro-retinol in pregnant mice, rats and rabbits following dosing with vitamin A, and (ii) the preferential accumulation of 14-hydroxy-4,14-retro-retinol in maternal and embryonic tissues, rather than in material plasma.

Nucleotide sequence of the gene coding for Clostridium botulinum (Clostridium argentinense) type G neurotoxin: genealogical comparison with other clostridial neurotoxins.

The neurotoxin gene from Clostridium botulinum type G was cloned as a series of overlapping DNA fragments generated using polymerase chain reaction (PCR) technology and primers designed to conserved regions of published botulinal toxin (BoNT) sequences. The 5'-end of the gene was obtained using a primer based on a conserved region of the nontoxic-nonhaemagglutinin gene lying upstream of the toxin gene. Translation of the nucleotide sequence derived from the cloned PCR fragments demonstrated that the gene encodes a protein of 1297 amino acid residues (rmm 149, 147). Comparative alignment of the determined BoNT/G sequence with those of other clostridial neurotoxins revealed highest sequence relatedness (approx. 58% amino acid identity) with BoNT/B of proteolytic and non-proteolytic C. botulinum. Tetanus toxin (TeTx) and other BoNT types revealed lower levels of relatedness with BoNT/G (approximate range 35-42% amino acid identity).

PCR cloning and nucleotide sequence determination of the 18S rRNA genes and internal transcribed spacer 1 of the protozoan parasites Cryptosporidium parvum and Cryptosporidium muris.

The genes encoding 18S rRNA and internal transcribed spacer 1 (1TS1) of Cryptosporidium parvum and Cryptosporidium muris were amplified from oocysts by PCR utilizing primers complementary to conserved regions of the 5' end of 18S and 5.8S rRNA. PCR products were cloned and the complete nucleotide sequences of two clones of each Cryptosporidium species were determined. The 18S rRNA genes of C. parvum and C. muris showed more than 99% sequence identity.

Diabetic embryopathy in C57BL/6J mice. Altered fetal sex ratio and impact of the splotch allele.

Maternal diabetes (types 1 and 2) induces a broad array of congenital malformations, including neural tube defects (NTDs), in humans. One of the difficulties associated with studying diabetic embryopathy is the rarity of individual malformations. In an attempt to develop a sensitive animal model for maternal diabetes-induced NTDs, the present study uses chemically induced diabetes in an inbred mouse model with or without the splotch (Sp) mutation, a putatively nonfunctional allele of Pax3. Pax3 deficiency has been associated with an increase in NTDs. Female C57BL/6J mice, either with or without the Sp allele, were injected intravenously with alloxan (100 mg/kg), and plasma glucose was measured 3 days later. A wide range of hyperglycemia was induced, and these diabetic mice were bred to C57BL/6J males, some carrying the Sp allele. Gestational-day-18 fetuses were examined for developmental malformations. Fetuses from matings in which either parent carried the Sp allele were genotyped by polymerase chain reaction. Maternal diabetes significantly decreased fetal weight and increased the number of resorptions and malformations, including NTDs. A significant correlation was found between the level of maternal hyperglycemia and the malformation rate. The sex ratio for live fetuses in diabetic litters was significantly skewed toward male fetuses. Matings involving the Sp allele yielded litters with significantly higher percentages of maternal diabetes-induced spina bifida aperta but not exencephaly, and this increase was shown to be associated with the presence of a single copy of the Sp allele in affected fetuses. Thus, Pax3 haploinsufficiency in this murine model of diabetic embryopathy is associated with caudal but not cranial NTDs.

Habitat and Vegetation Variables Are Not Enough When Predicting Tick Populations in the Southeastern United States.

Two tick-borne diseases with expanding case and vector distributions are ehrlichiosis (transmitted by Amblyomma americanum) and rickettiosis (transmitted by A. maculatum and Dermacentor variabilis). There is a critical need to identify the specific habitats where each of these species is likely to be encountered to classify and pinpoint risk areas. Consequently, an in-depth tick prevalence study was conducted on the dominant ticks in the southeast. Vegetation, soil, and remote sensing data were used to test the hypothesis that habitat and vegetation variables can predict tick abundances. No variables were significant predictors of A. americanum adult and nymph tick abundance, and no clustering was evident because this species was found throughout the study area. For A. maculatum adult tick abundance was predicted by NDVI and by the interaction between habitat type and plant diversity; two significant population clusters were identified in a heterogeneous area suitable for quail habitat. For D. variabilis no environmental variables were significant predictors of adult abundance; however, D. variabilis collections clustered in three significant areas best described as agriculture areas with defined edges. This study identified few landscape and vegetation variables associated with tick presence. While some variables were significantly associated with tick populations, the amount of explained variation was not useful for predicting reliably where ticks occur; consequently, additional research that includes multiple sampling seasons and locations throughout the southeast are warranted. This low amount of explained variation may also be due to the use of hosts for dispersal, and potentially to other abiotic and biotic variables. Host species play a large role in the establishment, maintenance, and dispersal of a tick species, as well as the maintenance of disease cycles, dispersal to new areas, and identification of risk areas.

Probiotics, prebiotics, and synbiotics: approaches for modulating the microbial ecology of the gut.

The microbiota of the human large intestine influences health and well-being. Whereas it has long been accepted that gut bacteria play a role in host pathogenesis, current opinion is that certain microflora components can have beneficial effects on gastroenteritis resistance, blood lipids, antitumor properties, lactose tolerance, and gastrointestinal immunity. It is postulated that in the infant gut an elevated bifidobacterial count may be associated with health advantages that breast-fed infants may have over formula-fed infants. Whereas beneficial aspects of the human gut flora still need definitive confirmation and mechanistic explanations, there is now interest in modulating the composition of gut flora such that a potentially more remedial community exists. This may be achieved through the targeted use of dietary supplementation. This article provides an overview of how probiotics, prebiotics, and synbiotics may contribute toward nutritional modulation of the gut microecology, with emphasis on the neonatal intestine where appropriate. The use of modern molecular methods, as an essential step forward for assessing the validity and accuracy of the modulatory approach, is also discussed.

Pax3 and the splotch mutations: structure, function, and relationship to teratogenesis, including gene-chemical interactions.

The current review focuses on the malformations resulting from mutations in Pax3 and the interactions of Pax3 mutations with chemically induced teratogenesis, as well as other mutant genes or genetic strains, as a paradigm to illustrate the connections among genetics, protein function, and teratology. Splotch mice result from various mutations involving Pax3, and Waardenburg syndromes I and III in the human are due to mutations in PAX3. The human and murine phenotype/genotype correlations are thus compared and contrasted. The role of Pax3 in normal development, as well as the regulation of Pax3 expression and DNA binding, are also addressed on the premise that a mechanistic understanding of normal developmental processes is prerequisite to full comprehension of the mechanisms by which abnormal development is induced. Pax3 encodes a transcription factor involved in myogenesis, melanogenesis and neurogenesis, as well as regulating genes that may be involved in other cellular processes. The primary goal of this review is to examine the role of a single important developmental gene in the interaction of genetics and abnormal development.

Pharmacokinetic determinants of embryotoxicity in rats associated with organic acids.

We have studied four organic acids of similar structure to further understand the basis of their developmental toxicity. Valproic acid (2-propyl pentanoic acid), ethylhexanoic acid, and octanoic acid are isomeric C8 organic acids but their teratologic potency varied widely. Valproic acid induced a moderate to severe teratologic outcome after a single oral administration of 6.25 mmoles/kg on day 12 of rat pregnancy. Twice as much ethylhexanoic acid (12.5 mmoles/kg) induced a less severe response. Octanoic acid was nonteratogenic even at the very high dose of 18.75 mmoles/kg. This latter result is undoubtedly due to poor intestinal absorption of octanoic acid, as the maternal plasma levels never reached half of those measured for valproic acid and ethylhexanoic acid. Moreover, only a tiny fraction of that in maternal plasma was actually transferred into the embryo. On the other hand, the peak concentration and duration of exposure to valproic acid and ethylhexanoic acid were very similar despite a more severe teratologic outcome following valproic acid, which indicated higher intrinsic activity of this latter agent. A fourth agent, methylhexanoic acid, was also studied and had no teratogenic effects when given at 14.1 mmoles/kg. Pharmacokinetic studies of this agent revealed higher peak concentrations in maternal plasma and embryo than valproic acid or ethylhexanoic acid, but the duration of exposure was shorter. We conclude that pharmacokinetic parameters can be important determinants of teratologic outcome and thereby help explain differing potencies of structurally similar chemicals.

Intrageneric relationships of Enterococci as determined by reverse transcriptase sequencing of small-subunit rRNA.

The 16S ribosomal ribonucleic acid (rRNA) sequences of eleven Enterococcus species were determined by reverse transcription in an attempt to clarify their intrageneric relationships. Comparative analysis of the sequence data revealed the presence of several species groups within the genus. The species E. avium, E. malodoratus, E. pseudoavium and E. raffinosus formed a distinct group as did E. durans, E. faecium, E. hirae and E. mundtii and the pair of species E. casseliflavus and E. gallinarum. Of the remaining species, E. cecorum, E. columbae, E. faecalis and E. saccharolyticus formed distinct lines of descent within the genus, whereas E. solitarius displayed a closer affinity with Tetragenococcus halophilus than with other enterococcal species.

Sequence of the gene coding for the neurotoxin of Clostridium botulinum type A associated with infant botulism: comparison with other clostridial neurotoxins.

The neurotoxin gene from a strain of Clostridium botulinum type A causing infant botulism was cloned as a series of overlapping polymerase chain reaction (PCR) fragments generated using primers designed to conserved regions of published botulinal toxin (BoNT) sequences. Translation of the nucleotide sequence derived from cloned PCR fragments demonstrated that the toxin gene encodes a protein of 1,296 amino acid residues. Comparative alignment of the derived infant BoNT/A sequence with those of other published neurotoxins revealed highest sequence relatedness with BoNT/A of classical food-borne botulism. The sequence identity between infant and classical BoNT/A was 94.9% for the light chain (corresponding to 23 amino acid changes) and 87.1% for the heavy chain (corresponding to 109 amino acid changes).

Studies on the large subunit ribosomal RNA genes and intergenic spacer regions of non-proteolytic Clostridium botulinum types B, E and F.

The large-subunit ribosomal ribonucleic acid (23S rRNA) genes of non-proteolytic (group II) strains of Clostridium botulinum toxin types B, E and F were amplified using the polymerase chain reaction (PCR), and cloned in Escherichia coli. Sequence determination showed that the 23S rRNA genes were 2910 nucleotides in length, and comparative analysis revealed approximately 99.5% sequence similarity. The 23S rRNA gene sequence of a strain phenotypically resembling non-proteolytic C. botulinum, except in not producing botulinal neurotoxin, was also determined and displayed 99.5% sequence similarity with those from toxigenic strains. A diagnostic sequence within the 23S rRNA characteristic for non-proteolytic C. botulinum was identified and used for the design of an oligonucleotide probe. Molecular hybridizations with PCR-amplified rDNA targets provided a precise and reliable method of identifying non-proteolytic (or Group II) C. botulinum and closely related non-toxigenic strains.

Phylogenetic analysis of Streptococcus saccharolyticus based on 16S rRNA sequencing.

Reverse transcriptase sequencing of 16S rRNA of Streptococcus saccharolyticus was performed in order to determine the phylogenetic position of this organism. On the basis of the sequence data Streptococcus saccharolyticus formed a distinct group with Enterococcus faecalis (type species of the genus Enterococcus) and other enterococcal species. Streptococcus saccharolyticus was found to be only distantly related to members of the genus Streptococcus sensu stricto. It is therefore proposed that Streptococcus saccharolyticus be reclassified in the genus Enterococcus, as Enterococcus saccharolyticus comb. nov.

Molecular taxonomic studies on some LL-diaminopimelic acid-containing coryneforms from herbage: description of Nocardioides fastidiosa sp. nov.

Molecular taxonomic studies were performed on two LL-diaminopimelic acid-containing coryneform isolates from herbage. The results indicate that the herbage strains represent a new species of the genus Nocardioides for which the name Nocardioides fastidiosa sp. nov. is proposed. The type strain is NCIB 12713.

Comparative analysis of 23S ribosomal RNA gene sequences of Bacillus anthracis and emetic Bacillus cereus determined by PCR-direct sequencing.

The primary structures of the 23S ribosomal RNA genes of Bacillus anthracis and an emetic strain of Bacillus cereus were determined by direct sequencing of enzymatically amplified chromosomal DNA. The 23S rRNA gene sequences of B. anthracis and B. cereus were found to be almost identical and showed only two differences (a single nucleotide change, and a single base insertion in B. cereus). The feasibility of using PCR-direct sequencing for the rapid sequence determination of large-subunit rRNA genes is demonstrated.

Comparative sequence analyses of the 16S rRNA genes of Lactobacillus minutus, Lactobacillus rimae and Streptococcus parvulus: proposal for the creation of a new genus Atopobium.

16S rRNA gene sequencing was performed on the species Lactobacillus minutus, Lactobacillus rimae and Streptococcus parvulus in order to clarify their taxonomic position. Based on comparative sequence analyses these organisms represent a hitherto unknown line of descent within the lactic acid group of bacteria for which a new genus, Atopobium gen. nov., is proposed.

Evidence for a close genealogical relationship between Afipia (the causal organism of cat scratch disease), Bradyrhizobium japonicum and Blastobacter denitrificans.

The primary structures of the small subunit rRNA of Bradyrhizobium japonicum and Blastobacter denitrificans were determined by direct sequencing of enzymatically amplified DNA. Comparative sequence analysis revealed that Bradyrhizobium japonicum and Blastobacter denitrificans are members of the alpha-2 subgroup of the Proteobacteria and show a very close phylogenetic relationship with the genus Afipia.

Phylogenetic analysis of some LL-diaminopimelic acid-containing coryneform bacteria from human skin: description of Propionibacterium innocuum sp. nov.

A new species, Propionibacterium innocuum, is proposed to accommodate strains of coryneform bacteria from human skin with phenotypic characters similar to those of the classical propionibacteria but differing in exhibiting primarily aerobic respiration and possessing a unique cell wall composition in which LL-diaminopimelic acid and arabinose occur together. The partial 16S rRNA sequence confirms an affinity with the genus Pro-pionibacterium and indicates that the species represents a distinct line within the genus. The type strain of Propionibacterium innocuum is NCTC 11082.

Molecular characterization of DNA encoding 23S rRNA and 16S-23S rRNA intergenic spacer regions of Aeromonas hydrophila.

Amplification of the gene encoding 23S rRNA of Aeromonas hydrophila by polymerase chain reaction, with primers complementary to conserved regions of 16S and the 3'-end of 23S rRNA genes, resulted in a DNA fragment of approximately 3 kb. This fragment was cloned in Escherichia coli, and its nucleotide sequence determined. The region encoding 23S rRNA shows high homology with the published sequences of 23S rRNA from other members of the gamma division of Proteobacteria. The sequence of the intergenic spacer region, between the 16S and 23S rRNA genes, was determined in five clones. Three types of spacer were identified: two clones were identical and encoded tRNA(Ile) and tRNA(Ala) while the remaining three clones contained tRNA(Glu), only two had the same spacer sequences. This variation in sequence indicates that the different clones may be derived from different ribosomal RNA operons.

A phylogenetic analysis of the genus Leuconostoc based on reverse transcriptase sequencing of 16 S rRNA.

The phylogenetic interrelationships of members of the genus Leuconostoc and some heterofermentative lactobacilli, which phenotypically resemble leuconostocs, were investigated by comparative analysis of their 16 S rRNA sequences. The six species, Leuconostoc mesenteroides, Leu. carnosum, Leu. citreum, Leu. gelidum, Leu. lactis and Leu. pseudomesenteroides exhibited a high degree of sequence similarity with each other and formed a phylogenetically coherent group, quite separate from all other lactic acid bacteria investigated. The species Leu. paramesenteroides was found to be phylogenetically distinct from the Leu. mesenteroides group of species and formed a natural grouping with the heterofermentative lactobacilli, Lb. confusus, Lb. kandleri, Lb. minor and Lb. viridescens. The rRNA sequence of the acidophilic species, Leu. oenos, displayed exceptionally low levels of homology with all of the other taxa examined. The 16 S sequence of Leu. oenos showed major nucleotide differences in relatively highly conserved positions of the molecule indicating this species is phylogenetically distinct and warrants a separate genus.

Analysis of DNA encoding 23S rRNA and 16S-23S rRNA intergenic spacer regions from Plesiomonas shigelloides.

Amplification of the gene encoding 23S rRNA of Plesiomonas shigelloides by polymerase chain reaction (PCR), with primers complementary to conserved regions of 16S and the 3' end of 23S rRNA genes, resulted in a DNA fragment of approximately 3 kb. This fragment was cloned in Escherichia coli and its nucleotide sequence determined. The region encoding 23S rRNA shows high homology with the published sequences of 23S rRNA from other members of the gamma division of Proteobacteria. The sequence of the intergenic spacer region, between the 16S and 23S rRNA genes, was determined in a further two clones. In one the sequence of a single tRNA(Glu) was found which was absent from the other two. This variation in sequence suggests that the different clones may be derived from different ribosomal RNA operons.